Cone photoreceptors are responsible for high acuity, central daylight and color vision. In humans there are 3 distinct subclasses of cone photoreceptors, each named for the specific wavelength of light to which they respond. Spectral sensitivity it mediated by the specific form of cone opsin that each cone subclass expresses. Cones that express S opsin respond to short wavelength light (blue: 420-440 nm) are referred to as “S” cones. Cones that respond to medium wavelength light (green: 534-545 nm) express M opsin and are referred to as “M” cones, and finally cones that express L opsin respond to long wavelength light (red: 564-580 nm) are referred to as “L” cones. Gene therapy based treatments for a number of diseases affecting cone photoreceptors are currently under development. One such disease, Achromatopsia, is characterized by an inability to see color, blindness in full sunlight (or at high light levels) and very poor visual acuity. Although congenital achomatopsia (ACHM) is a relatively rare disorder, it is a good target for gene therapy as the causative genes are known and proof-of-concept gene replacement studies in animal models have clearly shown success (Pang et al. (2010)). ACHM affects all classes of photoreceptors, including S cones. Recent evidence from case studies of patients with ACHM suggests that ACHM is progressive, with cones degenerating over time. Therefore, early intervention with a therapy that targets all cone photoreceptors would be ideal. Additionally, any disease that broadly affects cone photoreceptors, such as progressive cone dystrophy, would benefit from a gene therapy approach that was capable of targeting all cones.
In order to effectively and safely deliver genes to cone photoreceptors of ACHM affected individuals, gene therapy vectors must utilize promoters that meet the following criteria 1) the promoter must drive transgene expression both efficiently and selectively in cones, with no off-target expression in rod photoreceptors or other non-photoreceptor cell types, such as the retinal pigment epithelium (RPE), 2) the promoter must be capable of driving gene expression in all subclasses of cone photoreceptors, and 3) the promoter should be small, thereby allowing for sufficient carrying capacity of the vector to accommodate transgene DNA. To date, cone targeting promoters used in gene therapy proof-of-concept experiments of ACHM have been deficient in one or more of these criteria. In gene therapy studies by Alexander et al. (2007) and Komaromy et al. (2010) a 2100 base pair version of the human red/green opsin promoter (PR 2.1) was used to drive therapeutic transgene expression. In humans, the genes for M and L opsin are arranged in tandem on the X chromosome and therefore share a common promoter. In mouse, expression was limited to cones and some rod photoreceptors (Alexander et al. (2007)). In dogs, expression was limited to M and L cones (Komaromy et al. (2010)). While highly selective for M/L cone photoreceptors in dogs, PR2.1 mediated expression was not observed in S cones. Additionally the PR2.1 promoter is relatively large and in the case of the CNGB3 form of ACHM, AAV vectors (packaging size limitation of <5 KB) are barely able to accommodate promoter and cDNA, and this in turn reduces vector manufacturing efficiency. Promoters isolated from either the human or mouse blue cone (S) opsin gene have also been characterized for AAV mediate expression and gene replacement studies in ACHM animal models. These promoters are from nearly identical regions of the blue cone opsin genes of each respective species (i.e., are homologous). In rat, the human blue cone opsin promoter (HB569) drove reporter gene expression in all cone subclasses; however expression was weaker relative to the PR2.1 promoter (Glushakova et al. (2006)). In dog, HB569 performed poorly in terms of both specificity and efficiency, with relatively few L/M cones expressing transgene, rods and RPE positive for expression and overall weak expression. The mouse blue cone opsin promoter (mBP) has been tested in the context of gene replacement for the CNGA3 form of ACHM and performed well, however the likelihood is that like the closely related HB569, this promoter, will perform poorly in higher order mammals, such as dog and human. Finally, both human and mouse cone arrestin promoters have been utilized in AAV transduction experiments and later in gene replacement studies in ACHM animal models. As with the blue cone promoters, the human and mouse versions of cone arrestin promoters are homologues. In experiments performed in mice aimed at characterizing gene expression mediated by both the mouse cone arrestin promoter (mCAR) and the human cone arrestin promoter (hCAR), strong expression was observed. However specificity was poor, with rods and RPE clearly being transduced. In experiments utilizing mCAR that were performed in dog, the same general expression pattern was seen, with strong expression observed in all classes of cones and off-target expression in rods and RPE. Table 1 summarizes results of cone targeted promoter that have been used (to date) in AAV mediated gene delivery to the retina.
It has been our experience when utilizing photoreceptor specific promoters with AAV that specificity increases when moving from rodent (mouse and rat) to dog, and that the source organism from which the promoter sequence originated has little effect. In the few cases where photoreceptor promoters have been tested in primates, the results have been consistent with those obtained in dog. Therefore, in terms of predicting promoter activity in humans, we place emphasis on results obtained in dog experiments.
Ying et al. created a transgenic mouse line in which position −151 to +126 of human cone transducin alpha-subunit (GNAT2) gene was fused to chloramphenicol acetyltransferase gene followed by position −1622 to −1409 of the interphotoreceptor retinoid-binding protein (IRBP) gene (Ying et al. (1998)). Later, Ying et al. used the same general arrangement of elements to create a transgenic mouse line in which the goal was to ablate cone photoreceptors (Ying et al. (2000)). See FIG. 1 of Ying et al. (2000) for the arrangement of elements used by Ying et al. A resulting transgenic mouse line was characterized by Fong et al. and found to lack cone photoreceptors, and in ventral retina rod photoreceptors were also absent (Fong et al. (2005)). The region-specific absence of rod photoreceptors was reported as a consequence of developmental defect due to lack of cones. However, given that only 2.5% of photoreceptors are cones, loss of rods may have been due to mis-expression of the diphtheria toxin in rods.